RNA interference (RNAi) is an evolutionarily conserved cellular mechanism of post-transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to inhibit gene expression in a sequence-specific manner RNAi is controlled by the RNA-induced silencing complex (RISC) that is initiated by short double-stranded RNA molecules in a cell's cytoplasm. The short double-stranded RNA interacts with Argonaute 2 (Ago2), the catalytic component of RISC, which cleaves target mRNA that is complementary to the bound RNA. One of the two RNA strands, known as the guide strand, binds the Ago2 protein and directs gene silencing, while the other strand, known as the passenger strand, is degraded during RISC activation. See, for example, Zamore and Haley, 2005, Science, 309:1519-1524; Vaughn and Martienssen, 2005, Science, 309:1525-1526; Zamore et al., 2000, Cell, 101:25-33; Bass, 2001, Nature, 411:428-429; and, Elbashir et al., 2001, Nature, 411:494-498. Single-stranded short interfering RNA has also been shown to bind Ago2 and support cleavage activity (see, e.g., Lima et al., 2012, Cell 150:883-894). Importantly, the activity of single-stranded RNAi molecules should not be confused with that of single-stranded antisense RNA that inhibits translation of a complementary RNA in a stoichiometric fashion by base pairing to it, physically obstructing the translation machinery.
The RNAi machinery can be harnessed to destruct any mRNA of a known sequence. This allows for suppression (knock-down) of any gene from which it was generated, consequently preventing the synthesis of the target protein. Modulation of gene expression through an RNAi mechanism can be used to modulate therapeutically relevant biochemical pathways, including ones which are not accessible through traditional small molecule control. RNAi has also become a very important tool for target validation in the pharmaceutical industry.
Chemical modification of nucleotides incorporated into RNAi molecules leads to improved physical and biological properties, such as nuclease stability (see, e.g., Damha et al., 2008, Drug Discovery Today, 13:842-855), reduced immune stimulation (see, e.g., Sioud, 2006, TRENDS in Molecular Medicine, 12:167-176), enhanced binding (see, e.g., Koller, E. et al., 2006, Nucleic Acid Research, 34:4467-4476), and enhanced lipophilic character to improve cellular uptake and delivery to the cytoplasm. Thus, chemical modifications have the potential to increase potency of RNA compounds, allowing lower doses of administration, reducing the potential for toxicity, and decreasing overall cost of therapy.
While the sugar-phosphate backbone of most DNAs and RNAs are comprised of 3′-5′ internucleoside phosphodiester linkages, the physiochemical and biochemical properties of 2′-5′ linked ribonucleotides have been studied. Although not used for biological information storage, 2′-5′ linked oligoribonucleotides support Watson-Crick base pairing and are formed naturally during intron splicing and in interferon treated cells (see, e.g., Jim et al., 1993, Proc. Natl. Acad. Sci. USA, 90:10568-10572; Sawai et al., 1996, Biopolymers, 39:173-182; Premraj et al., 2002, Biophysical Chemistry, 95:253-272; Johnston and Torrence (1984) in Interferons: Mechanism of Production and Action, Vol. 3 (Friedman, R. M., Ed.), pp. 189-298, Elsevier, Amsterdam). There has been interest in using 2′-5′ linked oligoribonucleotides in antisense RNA applications as they exhibit the tendency to selectively hybridize with their RNA complements, rather than DNA complements (see, e.g., Hashimoto and Switcher, 1992, J. Am. Chem. Soc., 114:6255-6256; Dougherty et al., 1992, J. Am. Chem. Soc., 114:6265-6255), and display improved resistance toward several types of nucleases (see, e.g., Allul and Hoke, 1995, Antisense Res. Develop., 5:3-11; Kandimalla et al., 1997, Nucl. Acids Res., 25:370-378; Prakash et al., 1999, Bioorg. Med. Chem. Lett., 9:2515-2520). However, there has been only limited study of the impact of 2′-5′ linked ribonucleotides within RNAi oligonucleotides on the ability for such 2′-5′ linked oligoribonucleotides to appropriately and efficiently degrade target gene expression through an Ago2-mediated RNAi pathway.
Prakish et al. (2006, Bioorg. Med. Chem. Lett 16:3238-3240) reported on the activity in mammalian cells of siRNA duplexes that have 2′-5′ linked nucleotides. Results showed that an siRNA duplex comprising a 2′-5′ linked antisense strand and a 3′-5′ linked sense strand was not active in inhibiting mRNA expression, while an siRNA duplex with the reverse composition (i.e., a 3′-5′ linked antisense strand and a 2′-5′ linked sense strand) was active. They concluded that 2′-5′ linkages are tolerated in the sense strand of siRNA duplexes but not in the antisense strand. Since the 5′-end of the antisense strand, in particular, is important for loading siRNA into RISC, positioning nucleation with mRNA and subsequent cleavage, the authors provide that it is likely crucial for the 5′ end of the antisense strand to adopt correct geometry in order to appropriately interact with RISC. They conclude that 2′-5′ internucleoside linkages at the 5′ end of the antisense strand, thus, may not be capable of adopting the proper conformation to support that interaction.
PCT International application serial no. PCT/US2011/033961, published as WO 2011/139699 on Apr. 26, 2011, discloses 5′ modified nucleosides and oligomeric compounds incorporating the modified nucleosides. The 5′ modified nucleosides disclosed are preferably located at the 5′ terminus of an oligonucleotide and have modifications at the 5′ carbon of the sugar moiety of the nucleoside and, optionally, additional modifications at the 2′ carbon. The 5′ modified nucleosides disclosed in PCT/US2011/033961 are linked to an adjacent nucleoside by a traditional 3′-5′ internucleoside linkage.